Sunil Vasu

Fluorescence of hydrogel film can be measured. See how it was done.

Article Polymer composite fluorescent hydrogel film based on nitroge...

https://www.spiedigitallibrary.org/conference-proceedings-of-spie/5323/0000/Measuring-tear-protein-mobility-in-thin-hydrogel-films-with-fluorescence/10.1117/12.529758.short

Regarding fluorescence assay using 96 well plate, shall I use a plate has wavelength range includes the emission wavelength.

Yes, the fluorescence of a hydrogel film can be measured by cutting it into small pieces and placing it in a 96-well plate sample holder, but there are a few key considerations to ensure accurate measurements:

1. Hydrogel Piece Size and Placement:

• Uniform Size: The pieces of hydrogel film should be cut into uniform sizes to ensure consistency in fluorescence intensity across wells. Variations in size can lead to uneven distribution of the fluorescent signal.
• Even Distribution: Ensure that the hydrogel pieces are evenly distributed at the bottom of each well. In some cases, using a transparent or optically clear 96-well plate might help improve signal detection.

2. Fluorescent Dye or Molecule Concentration:

• If the hydrogel is embedded with a fluorescent dye or contains fluorescent molecules, the concentration of these fluorophores needs to be uniform within the hydrogel to avoid variability in signal intensity.

3. Measurement Conditions:

• Solvent Consideration: If the hydrogel requires a specific hydration level or solvent environment to maintain its fluorescence properties, ensure that the hydrogel pieces are in the appropriate conditions (e.g., immersed in buffer or water) inside the wells.
• Excitation and Emission Settings: Use a plate reader that allows you to specify the correct excitation and emission wavelengths for the fluorescent material being studied. Ensure that the optical path from the excitation source and the detector is aligned properly for fluorescence measurement.

4. Potential Issues:

• Thickness and Opacity: If the hydrogel pieces are too thick or highly opaque, they might scatter or absorb some of the excitation or emission light, leading to inconsistent measurements. Thinner films or thinner pieces of hydrogel may yield more accurate results.
• Auto-fluorescence: Some hydrogels can exhibit auto-fluorescence, which might interfere with the detection of the desired fluorescent signal. A control sample (without the fluorophore) should be measured to account for any background fluorescence from the hydrogel itself.

5. Alternatives:

• If the hydrogel film is large or difficult to handle in pieces, another option could be using a flatbed fluorimeter where the entire hydrogel film can be placed in the measurement path without cutting, avoiding any uneven distribution or size issues.